PROJECT SUMMARY/ABSTRACT The endoplasmic reticulum (ER) orchestrates protein folding and secretion and the unfolded protein response (UPR) maintains ER homeostasis. The UPR is mediated by three signal transduction pathways, controlled by the proteins PERK, IRE1?, and ATF6, that act to clear unfolded proteins from the ER. Sustained elevation of the UPR, or its insufficient activation, contributes to the pathology of diseases such as diabetes, obesity, atherosclerosis, and cancer. Osteoblasts are bone forming cells that secrete large amounts of collagen and have extensive ER. Moreover, humans and mice lacking a functional version of the PERK gene display several metabolic defects and have low bone mass. However, it is not clear whether the reduced bone mass is due to a decrease in the UPR in osteoblasts or in other cell types. Some osteoblasts become buried in new bone matrix and become osteocytes, which produce factors that control bone remodeling. Osteocyte death can induce factors that stimulate production of bone resorbing osteoclasts. In preliminary studies, we found that induction of ER stress in mice and osteocyte-containing bone cultures increased osteoclastogenic cytokine production, osteoclast number, and bone resorption. Conversely, in a mouse model of glucocorticoid excess, which causes bone loss in humans, we observed reduced expression of downstream mediators of the UPR. This evidence suggests that either excessive or insufficient UPR activation can lead to bone loss. Based on this, we propose the central hypothesis that components of the UPR play an essential role in osteoblast differentiation and function, and dysregulation of the UPR in cells of the osteoblast lineage contributes to pathological bone loss. To test this hypothesis, we will determine whether Perk plays an essential role in osteoblast physiology by conditionally deleting this gene from cells of the osteoblast lineage using Osx1-Cre transgenic mice (Aim 1). We will also determine the skeletal consequences of sustained elevation of the UPR in cells of the osteoblast-lineage by deleting the protein-folding co-chaperone ERdj4 specifically in these cells followed by a comprehensive analysis of the skeleton and osteoblast gene expression (Aim 2). Lastly, we will determine whether glucocorticoid excess inhibits bone formation in mice, in part, by suppressing UPR activation using UPR reporter mice treated with exogenous glucocorticoids and biochemical analyses of UPR pathways (Aim 3). The scientific theme of the proposed Center for Musculoskeletal Disease Research is that genetic analysis of musculoskeletal diseases will support development of novel therapies for such conditions. The proposed studies are aligned with this theme and their successful completion may identify new therapeutic targets for common causes of osteoporosis. These studies will be supported by all three proposed COBRE research cores and the Project Leader will be mentored by a two-member team and other members of Center. Results produced during the first 2 years of the project will lead to the submission of an R01 grant application by the end of the second year of COBRE support.